Lactose Intolerance is an increasingly prevalent malady that affects 20% of the American population, 90% of African Americans, 95% of Asian Americans, and 99% of Native Americans. Current treatments involve the treatment of milk and milk products with the enzymes that commonly break down lactose into its component sugars. Due to the origin of the [unreadable]-galactosidase enzyme most commonly employed - a mesophilic bacteria known as Aspergillus oryonzae the enzyme is ill suited to function at the low temperatures that milk and milk products are typically stored at. Use of psychrophilic, or cold loving, bacteria as a source for [unreadable]-galactosidases has had its own problems as well. These enzymes are usually extremely thermolabile and unable to withstand the temperatures encountered during the flash pasteurization process. The project detailed here, provides a strategy not only to engineer an enzyme that is stable to high temperatures and capable of significant activity over a broad range of temperatures, but also tests a number of novel paradigm shifts in understanding of the process of temperature adaptation and thermostability. With a large amount of preliminary data to suggest high chances of success, this project bridges the gap between a truly random mutagenesis approach to protein design and basic science research that promises to elucidate some of the underlying fundamental concepts governing protein adaptation to temperature and the nature of thermostability and protein fold specificity. The eight stranded a/[unreadable] (TIM) barrel protein fold is the largest of protein folds, comprising over 10% of all known protein structures. This family also has the largest known distribution of catalytic mechanisms, substrate specificities, and chemical functions. Proposed here are experiments to elucidate "Fold Specific" adaptation strategies while engineering a new, and therapeutically useful enzyme. These strategies could potentially be applicable to this entire family of proteins giving it significance to academia, industry, and medicine. This project will entail extensive use of molecular biology techniques, biochemical characterization of enzymes, use of "Directed Evolution" techniques and a variety of bioinformatics approaches to understanding protein structure-function relationships. In the process two new methods are developed to understand, quantify, visualize, and communicate complicated conformational information while at the same time yielding information with predictive value that may be directly applied to a wide variety of proteins even outside of the fold. Lastly, this project Design also leverages departmental funds for supplies and human effort by incorporating a significant aspect of the research into junior and senior level Biochemistry Lab courses. PUBLIC HEALTH RELEVANCE: This project will develop a therapeutic agent for individuals with lactose intolerance, a malady that affects over 20% of Americans, 90% of African Americans, 95% of Asian Americans, and over 99% of Native Americans. At the same time this project will create new paradigms to aid in methods of drug discovery and design.